Precise regulation of smooth muscle cell (SMC) growth, migration, and differentiation is necessary for proper vascular development, and defective control of these processes contributes to the progression of several prominent congenital and adult onset cardiovascular diseases. Extensive studies indicate that these SMC functions are regulated by growth factors and extracellular matrix (ECM)-integrin interactions and that activation of the non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) plays a critical role in these signaling pathways. We have previously demonstrated that an endogenous inhibitor of FAK, termed FRNK, is selectively expressed in SMC with particularly high levels observed in conduit blood vessels and that FRNK expression is dramatically up-regulated during post-natal vascular development and following vessel injury. These studies suggested that integrin matrix signaling in SMC was unique and that precise regulation of FAK activity was critical during vascular development and vessel injury repair. Indeed we have demonstrated that conditional inactivation of FAK (by homologous recombination) in wnt-1 or nkx2.5-derived SMC led to persistent truncus arteriosus that was incompatible with post-natal life. Since aorticopulmonary septation involves dynamic control of several SMC processes, we have continued to study the role of FAK and FRNK in SMC using a variety of loss/gain of function approaches. Inhibition of FAK activity by genetic deletion had little effect on cell growth or ERK activation. However, it strongly inhibited PDGF-BB-mediated cell polarization and migration, an effect likely due to defective activation of the small GTPase Rac-1. Interestingly, results from our in vitro and in vivo models also indicated a strong inverse correlation between FAK activity and SMC differentiation (as assessed by SMC differentiation marker gene expression). In an attempt to further delineate the FAK-dependent mechanisms involved in these responses, we identified the LIM domain adapter protein, leupaxin, in a yeast two-hybrid screen for FAK interacting proteins expressed in SMC. We utilized siRNA-mediated approaches to deplete leupaxin from SMC and these studies revealed that leupaxin was essential for SMC chemotaxis. We also made the interesting and potentially important discovery that leupaxin shuttles between focal adhesions and the nucleus and that this process was regulated by FAK signaling. We also demonstrated that ectopic expression of leupaxin up-regulated multiple SMC differentiation marker genes; that knock-down of leupaxin attenuated SMC differentiation marker gene expression; that leupaxin interacted with the SM a-actin promoter in vivo; and that leupaxin interacted physically and functionally with SRF and the powerful SRF co-factor, myocardin. In this proposal we seek to determine how FAK regulates SMC phenotype during vascular morphogenesis and to identify the precise mechanism(s) by which FAK and leupaxin alter SMC motility and differentiation. We hypothesize that leupaxin serves to integrate these diverse SMC functions and regulates migration by targeting Rac-1 activation to the leading edge of motile cells and differentiation by regulating the formation of a functional SRF transcription factor complex on SMC differentiation marker gene promoters.